Parasites of the trypanosomatid protozoan genus Leishmania are responsible for a spectrum of tropical diseases that afflict more than 10 million people worldwide, and depending on the specific speciesand mmune status of the infected person, can be severe or fatal. In several countries, leishmaniasis is a common opportunistic infection in AIDS patients, and many USsolidiers stationed in the Middle East have been infected. The goal of our research is to develop and apply new methods for the identification of genes used by this parasite to carry out its infectious cycle. Our premise is that the availability of powerful molecular genetic tools, and identification of genes important to parasite virulence, will radically advance our ability to develop improved control strategies. In previous studies methods were developed for manipulating the parasite genome in a variety ofways by forward or reverse genetics, and to develop functional genomic approaches such as transposon mutagenesis and expression profiling. Success with expression profiling now enables us to incorporate this powerful methodology more comprehensively into our research program, and undertake a broader survey of patterns and mechanisms of gene expression in Leishmania, making use of both genetic and biological methodological advances from the prior grant period. The goal is to identify genes showing patterns of expression suggestive of functional roles in the infectious cycle, for example stage-specific expression or alterations in response to relevant stimuli, whose predictions and implications can then be tested experimentally. One example that will be pursued involves a new perspective on the differentiation of the parasite into the infectious metacyclic form transmitted by sand flies, involving global changes in parasite gene expression probably mediated by changes in chromating structure, specific changes in expression arising from a combination of mechanisms, and biological changes involving massive cellular remodeling and 'shrinkage'possibly mediated by autophagy.